This invention relates to a method and apparatus for measuring the degree of an antigen-antibody reaction in order to determine the antigen, antibody or hapten level. More specifically, the invention relates to an immunological quantitative method and apparatus in which use is made of a suspension of insoluble microscopic carrier particles having antigen or antibody molecules bonded thereto, wherein the agglutination reaction among the carrier particles based on the antigen-antibody reaction is measured by optical means. Alternatively, the immunological quantitative method and apparatus to which the invention pertains are adapted to optically measure the degree of a so-called agglutination inhibiting reaction. This is a reaction in which a specimen of interest is introduced, along with an antibody, into a suspension of insoluble microscopic carrier particles having antigenic molecules bonded thereto. The introduced antibody forms bonds with the antigen molecules borne by the carrier particles, causing the aggregation of the carrier particles. Such aggregation is inhibited by the free antigen attributed to the specimen, so that measuring the agglutination inhibiting reaction makes it possible to quantitatively determine the antigen level of the specimen.
The in vitro analysis of substances, beginning with immunological analytical methods which utilize the high degree of specificity between antigens and antibodies, is an important technique essential to modern medical diagnosis and treatment. Radioimmunoassay (RIA) and enzyme immunoassay (EIA) methods are available for the in vitro immunological analysis of small quantities of substances. The RIA method, however, requires special facilities and trained technicians for the handling of radioactive isotopes, and an attendant problem is the treatment of wastes following analysis. In addition, the RIA method is disadvantageous in that it requires the implementation of a complex operation known as B/F separation, in which an antibody with a bonded antigen (or an antigen with a bonded antibody) is separated from a free antibody (or free antigen). The EIA technique does not employ radioactive material, but does generally call for the B/F separation. A homogeneous immunoassay technique does exist in which B/F separation is unnecessary. With this technique the antigen is bonded to an enzyme, and it is required to maintain the antigenic property of the antigen molecules as well as the activity of the enzyme. When the antigen molecules are as massive as protein molecules, however, enzyme activity is difficult to maintain, so there is a restriction upon the kind of antigen molecules to which this method can be applied.
In recent years, various methods based on the quantification of agglutination reactions using an insoluble carrier have been proposed for the purpose of detecting antigen or antibody molecules. Such methods include analysis based on electrical resistance pulses, light scattering and turbidimetry. For example, Philip Blume and Leonard J. Greenberg describe a method based on light scattering (Clinical Chemistry, Vol. 21, No. 9, 1975, pp. 1234-1237.). Utilizing the fact that the angular dependence of scattered light intensity varies with the particle size of a particle suspension, the authors find the angular differential (tangential slope) of the scatter intensity at a constant angle and establish a relation between the value found and the concentration of a specimen of interest in order to assay a rheumatoid factor. Latex particles are employed to effect the agglutination reaction. In addition, U.S. Pat. No. 4,174,952 issued to David S. Cannel et al., as well as the periodical Molecular Immunology (Vol. 17, 1980, pp. 81-92), discloses determining the degree of agglutination reactions by measuring the ratio of intensities of light scattered at angles of 90.degree. and 10.degree. with respect to the path of the incident light. According to the disclosed methodology, an agglutination reaction gives rise to an increase in average particle diameter and a simultaneous decrease in the total number of particles. The intensity of light scattered at 90.degree. therefore will decrease as agglutination proceeds, while the scatter intensity at 10.degree. will increase. This permits the ratio of these two intensities to be correlated to the degree of the agglutination reaction. Further, the periodical Immunochemistry (Vol. 12, 1975, pp. 349-351; Vol. 13, 1976, pp. 955-962), as well as U.S. Pat. No. 4,080,264 (issued in 1978), discloses that when a specimen is irradiated with laser light, (a) the scattered light receives a Doppler shift, and has its spectrum line width broadened, owing to the Brownian motion of the insoluble carrier particles, and (b) that it is possible to analyze very small quantities of IgA or human chorionic gonadotropin by finding the diffusion constant of the Brownian motion from the half-value width of the above spectrum. The basic principles involved here are optical mixing spectroscopy or light beating spectroscopy. Light beating spectroscopy provides a high level of sensitivity since detection is possible even at the stage where the carrier particles are in the form of microscopic polymers before growing into large visible or microscopic clumps. In order to analyze the frequency of the output current from the photodetector, however, light beating spectroscopy requires the installation of costly measuring instruments such as a spectrum analyzer or correlator, as well as considerable skill in operation and maintenance.
A basic arrangement for light beating spectroscopy is as shown in FIG. 1. A laser beam of a fixed wavelength emitted by an He-Ne, Ar-ion or other laser 1 is focused by a lens 2 and irradiates a scattering cell 3 containing the specimen of interest. Light scattered through an angle .theta. with respect to the optic axis of the laser beam is detected by a photodetector 6, such as a photomultiplier tube, after passing through slits, 4, 5 which determine the field of view. The output signal from the multiplier tube 6 is subjected to a frequency selection operation by a spectrum analyzer 8 after amplification by a preamplifier 7. The signal then is applied to a recorder 10 via a squaring circuit 9 to provide an indication of the frequency spectrum.
Before proceeding further, let us discuss the light scattering phenomenon exhibited by an aggregate of insoluble carrier particles that makes possible the application of the foregoing principle. Monodisperse latex particles suspended in a liquid exhibit Brownian motion. The following well-known formula may be used to express the spectral distribution of scattered light intensity arising from the translational Brownian motion of spherical particles: ##EQU1## Where .DELTA.v: frequency difference between incident and scattered light
D: diffusion constant of particles PA1 K: wave vector given by ##EQU2## (.mu..sub.o : wavelength of incident light in vacuum; n.sub.o : refractive index of solvent; .theta.: scatter angle) PA1 kB: Boltzmann constant, PA1 T: absolute temperature, PA1 d: particle diameter, PA1 .eta.: solvent viscosity coefficient.
For spherical particles, the Stokes-Einstein relation gives us: EQU D=(kBT/3.pi.d.eta.) (2)
where
Since Eq. (1) may be written I(K. .DELTA.v).varies.2/K.sup.2 D at the center frequency, namely for .DELTA.v=0, the beating signal has the form of a Lorentz equation, and the half-value width .DELTA.V.sub.1/2 may be written: EQU .DELTA.v.sub.1/2 =K.sup.2 D (3)
for .DELTA.v satisfying I(K, .DELTA.v)=1/K.sup.2 D. Thus, if we measure the half-value width of the frequency spectrum of the beating signal arising from the scattered light, then the diffusion constant D can be computed since K is determined by the values of .lambda..sub.o, n.sub.o and .theta..sub.o, which are already known. When aggregation occurs among carrier particles, the aggregate usually is rod-shaped or elliptical rather than spherical owing to the particle alignment or internal structure. Since such aggregates possess rotational freedom, however, the agglutination reaction can be measured quantitatively on the basis of an increase in the diameter of the aggregates. In actual practice, however, not only is the number of monomers (particles) in each aggregate not fixed, but there are also differences in size and a wide variety of alignments, internal structures and particle distributions. A theoretical analysis of the above is attempted in the periodical Bulletin of Mathmatical Biology (Vol. 42, pp. 17-36 and 37-56).
In summary, with light beating spectroscopy, the increase in particle diameter due to the agglutination reaction is detected as an average value taken among the aggregates. Thus, factors remain which prevent an improvement in measurement accuracy and sensitivity. One such factor obviously is the distribution of the degree of aggregation, and another is that it is not possible to gain a full understanding of the reaction sytem at the beginning of the agglutination reaction.